Stable cell lines expressing herg

ABSTRACT

A stable eukaryotic cell line that expresses hERG and exhibits a stable current under electrophysiological test conditions is provided.

RELATED APPLICATIONS

This application claims priority from U.S. Ser. No. 60/453,015, filedDec. 21, 2005, and U.S. Ser. No. 60/841,965, filed Sep. 1, 2006, bothincorporated herein by reference in full.

FIELD OF THE INVENTION

This invention relates generally to stable cell lines that express thevoltage-gated hERG potassium channel, and methods of using said cells totest compounds for their ability to inhibit hERG current.

BACKGROUND OF THE INVENTION

Ion channels constitute a relatively small class of pharmaceuticaltargets, in part because ion channel screening assays have beendifficult to automate and format for high throughput. However, recentadvances in electrophysiology (P. B. Bennett et al., Trends in Biotech(2003) 21(12):563-69; C. Wood et al., Drug Disc Today (2004)9(10):434-41) have rekindled interest in ion channels as targets fordrug discovery. A number of ion channels have been linked to inheriteddiseases, leading to the study of ion channel modulators for thetreatment and prevention of disease (D. Owen et al., Drug Disc World(2002) 48-61).

HERG is an ion channel of particular interest to the pharmaceuticalindustry, although as a safety/toxicology problem rather than a targetfor developing modulators (ICH S7B Guidance for Industry, October 2005;J. I. Vandenberg et al., Trends Pharm Sci (2001) 22(5):240-46). Thevoltage-gated hERG potassium channel contributes to therapidly-activating delayed rectifier potassium current (IKr) of thecardiac action potential. Drug interaction with the hERG potassiumchannel has been implicated in electrocardiogram QT intervalprolongation and the cardiac arrhythmia known as Torsades de Pointes(“TdP”; see C. E. Chiang and D. M. Roden, J Am Coll Cardiol (2000)36(1):1-12.; D. M. Roden, N Eng J Med (2004) 350:1013-22.). TdP can befatal, and the risk of inducing it has led to withdrawal andnon-approval of pharmaceutical products.

High throughput screening of drug candidates to determine their possibleeffect on hERG has proven to be difficult, based in large part on theunavailability of a stable cell line that expresses hERG at sufficientsurface concentrations, and is a suitable subject for high throughpution flow measurement instruments.

SUMMARY OF THE INVENTION

We have now invented hERG expressing cell lines that reproducibly formedstable seals with large current amplitudes using standard and automatedpatch clamp set ups. The cell lines of the invention produce IC₅₀ valuesthat are representative of those reported in the literature usingnon-high-throughput methods.

One aspect of the invention is a stable eukaryotic cell line thatexpresses hERG, and is capable of exhibiting a test current that variesby less than approximately 20% peak current amplitude under controlconditions for over one hour.

Another aspect of the invention is a method for determining thepropensity of a test compound to inhibit hERG conductance activity, bycontacting a cell of the invention with said test compound, measuring atest current under electrophysiological conditions, and determining ifthe test current is lower in the presence of the test compound.

DETAILED DESCRIPTION OF THE INVENTION

All publications cited in this disclosure are incorporated herein byreference in their entirety.

Definitions

Unless otherwise stated, the following terms used in this Application,including the specification and claims, have the definitions givenbelow. It must be noted that, as used in the specification and theappended claims, the singular forms “a”, “an,” and “the” include pluralreferents unless the context clearly dictates otherwise.

“Agonist” refers to a compound that enhances the activity of anothercompound or receptor site.

“Antagonist” refers to a compound that diminishes or prevents the actionof another compound or receptor site.

The term “drug candidate” refers to a compound or preparation which isto be tested for possible effect in the treatment of a disease state inan animal, regardless of whether said drug candidate has any knownbiological activity.

The term “homologous” as used herein refers to a protein that performssubstantially the same function in another subject species and sharessubstantial sequence identity, to the extent that they are recognized inthe art as being different versions of the same protein, differingprimarily in the species in which they are found. Thus, for example,human ERG, mouse ERG, and rat ERG are all considered homologous to eachother.

“Modulator” means a molecule that interacts with a target. Theinteractions include, but are not limited to, agonist, antagonist, andthe like, as defined herein.

“Disease” and “Disease state” means any disease, condition, symptom,disorder or indication.

The term “cell line” refers to a clone of immortalized mammalian cells.A “stable” cell line is a cell line that exhibits substantiallyconsistent characteristics over time (e.g., with each doubling). Astable cell line within the scope of this invention provides asubstantial proportion of cells that are capable of providing a sealresistance of greater than about 50 MOhm, a current amplitude of greaterthan about 200 pA, and provide a current amplitude that does not vary bymore than approximately 20% over one hour under control conditions.

The terms “progeny” and “descendents” as used herein refers to cellsobtained by culturing or otherwise growing a cell of the invention.

The term “derivative” as used herein refers to a cell that is obtainedby modifying, fusing, transfecting, transforming, or otherwise changinga cell of the invention. For example, derivatives can be created bytransfecting a cell of the invention with a plasmid or virus, by fusingit to a hybridoma cell, and the like.

The term “electrophysiological measurements” or “patch clamp experiment”refer to an experimental procedure in which the voltage potential ofpart or all of a cell membrane (typically in an isolated cell) ismaintained at a predetermined voltage, then subjected to one or morechanges in voltage, during and/or after which the current passingthrough the membrane is measured. In the hERG measurement experimentused herein, a cell expressing hERG on its surface is first voltageclamped at a holding potential of −80 mV, leak subtraction wascalculated from a 100 msec pulse to −40 mV, followed by 1000 msec at 20mV (prepulse), and 500 msec at −40 mV (test pulse). hERG current wasmeasured as the peak (beginning of the test pulse) at −40 mV aftercorrection with leak current. Variations of this protocol can beapplied. The hERG current inhibition due to drug interaction with thehERG potassium channel is measured during the test pulse and is noted as“test current”. In cell lines of the invention, the test current variesby less than approximately 20% in a control situation, lasting up to onehour. The term “patch clamp apparatus” refers to any instrument ordevice suitable for conducting such measurements, such as, for example,standard patch clamp, an IonWorks HT, IonWorks Quattro, PatchXpress7000A and the like.

“Subject” includes mammals and birds. “Mammals” means any member of themammalia class including, but not limited to, humans; non-human primatessuch as chimpanzees and other apes and monkey species; farm animals suchas cattle, horses, sheep, goats, and swine; domestic animals such asrabbits, dogs, and cats; laboratory animals including rodents, such asrats, mice, and guinea pigs; and the like. The term “subject” does notdenote a particular age or sex.

“Treating” or “treatment” of a disease state includes (i) preventing thedisease state, i.e. causing the clinical symptoms of the disease statenot to develop in a subject that may be exposed to or predisposed to thedisease state, but does not yet experience or display symptoms of thedisease state; (ii) inhibiting the disease state, i.e., arresting thedevelopment of the disease state or its clinical symptoms; or (iii)relieving the disease state , i.e., causing temporary or permanentregression of the disease state or its clinical symptoms.

All patents and publications identified herein are incorporated hereinby reference in their entirety.

General Method

The invention provides cell lines that express hERG and are suitable foruse in automated, high throughput electrophysiology assays, and methodsfor using such cells to screen compounds for potential hERG inhibitoryactivity.

The cell lines of the invention are designed for use in planar patchelectrophysiology systems by virtue of the fact that they are adapted togrowth in suspension. They have been used on systems like the IonWorksHT planar patch system and the PatchXpress 7000A, but may be used inother devices or systems as well.

Cell lines can be cultured in suspension using Ex-cell 301 (JRH, CatJRH-14331), 10% Fetal Bovine Serum (Gibco, Cat 16140-089) and 0.25 mg/mlGeneticin (Gibco, Cat 10131-035). Cells are preferably grown at 35±2 C,supplemented with 5% CO₂, in 50-100 ml volumes in 1 liter shake flasks,at 90-100 rpm (2 inch shaker amplitude). For optimal performance, celltiters are kept between about 10⁵ and about 10⁶ cells/ml.

Expression of hERG can be verified by standard methods, for example byWestern blot after cell lysis. Expression of hERG current can varydepending on cell culture conditions.

The stability of a cell line can be assessed using a standard patchclamp method, by clamping cells obtained from the cell line, pulsingthem, and measuring the resulting currents at a plurality of timepoints. In a stable cell line of the invention, the current does notvary by more than 20% in one hour under control conditions.

For adherent cells, removal usually requires the use of a dissociationreagent, such as culture medium supplemented with trypsin or Versene™.For suspension adapted cells, no dissociation reagent is typicallyrequired. Cells are resuspended in the electrophysiology recordingsolution prior to experimental use.

HERG current measurements are conducted on the cells of the invention inthe presence and absence of test compounds. For screening purposes, itis sufficient to note the concentration at which the test compoundinhibits the hERG current by about 50% or greater.

In the practice of the methods of the invention, cells from a cell lineof the invention are contacted with or exposed to test compounds,optionally including positive and negative control compounds, and thedegree to which hERG activity is inhibited is measured by determiningthe effect (if any) on current during electrophysiological measurements.Thus, for example, one can apply cells of the invention to a patch clampapparatus substrate, and contact individual cells with a test compound.The test compounds may be used in a plurality of concentrations, or mayall be used at a predetermined concentration (for example, 10 μM, 20 μM,50 μM, and the like). Compounds are typically dissolved inelectrophysiological recording solution. The cells are then patchclamped and pulsed as described herein, and the test current detected.Compounds that cause a substantial decrease in test current areconsidered to inhibit hERG activity at that concentration. Preferably,the test current is compared against one or more controls, which may bethe same cells of the invention prior to application of the testcompounds, or may be substantially identical cells (for example, derivedfrom the same cell culture) and subjected to positive and/or negativecontrol compounds.

Utility

The cell lines of the invention are useful for providing cell-surfaceexpression of hERG in stable yield, and serve as suitable substrates forhigh throughput hERG activity screening using electrophysiologicalmethods. Thus, using the cell lines of the invention, one can screendrug candidates quickly and efficiently for their possible interactionwith hERG. Methods of the invention are useful for high throughputscreening of drug candidates and other compounds, to determine theirinteraction and/or modulation of hERG, and thus an element of theirpotential cardiotoxicity.

EXAMPLES

The following preparations and examples are given to enable thoseskilled in the art to more clearly understand and to practice thepresent invention. They should not be considered as limiting the scopeof the invention, but merely as being illustrative and representativethereof.

Cell culture media components included Ex-cell 301 (JRH, Cat JRH-14331),10% Fetal Bovine Serum (Gibco, Cat 16140-089) and 0.25 mg/ml Geneticin(Gibco, Cat 10131-035). Cells are grown at 35±2 C, supplemented with 5%CO₂, in 50-100 ml volumes in 1 liter shake flasks, at 90-100 rpm (2 inchshaker amplitude). For optimal performance, cell titers are kept between10 ⁵ and 10 ⁶ cells/ml.

Electrophysiology recording solutions for both standard and automatedpatch clamp (PatchXpress 7000A) include Internal Buffer (in mM, fromSigma unless otherwise noted): 140 KCl (Cat P-9541), 6 EGTA (CatE-3889), 5 Hepes (Cat H-3784), 5 MgCl₂ (Cat M-1028), 5 ATP-Na₂ (CatA-2383) pH 7.2 with KOH (J.T. Baker, Cat 3143-01); External Buffer (inmM, from Sigma unless otherwise noted): 150 NaCl (Cat S-3014), 10 Hepes(Cat H-3784), 4 KCl (Cat P-9541), 1.2 CaCl₂ (Cat C-3306), 1 MgCl₂ (CatM-1028), pH to 7.4 with HCL (J.T. Baker Cat 5619-02). Patch Substratesinclude PatchPlates™ (Cat 9000-0688) and SealChips™ (1-SealChip16-K)distributed by Molecular Devices Corp.

Electrophysiology: Voltage pulse protocol: holding potential was −80 mV,leak subtraction was calculated from a 100 msec pulse to −40 mV,followed by 1000 msec at 20 mV (prepulse), and 500 msec at −40 mV (testpulse). hERG current was measured as the peak (beginning of the testpulse) at −40 mV after correction with leak current.

IC₅₀ Curve Generation and Statistics: Concentration-response curves arefitted in Excel Fit (version 3, ID-BS) with the Four ParameterLogistical Model or Sigmoidal Dose Response Model, Equation 205.Fractional inhibition(I_(Compound)/I_(Control))=1/(1+[Compound]/IC₅₀)^(n) ^(H) , where I iscurrent, IC₅₀ is the concentration of compound required to inhibitcurrent by 50% and n_(H) is the Hill coefficient.

Example 1 CHO-K1 Cell Line Expressing hERG

(A) A CHO-K1 cell line stably expressing high levels of functional hERGchannels was generated as follows. First, wild type CHO-K1 cells weretransfected with a plasmid encoding a (CMV-promoter-cyan fluorescenceprotein-IRES-hygromycin resistance marker) cassette. Two nonidenticalloxP sites are located on this construct, one between the CMV-promoterand the cyan fluorescence protein ORF, and one at the 3′-end of thehygromycin resistance marker. From random CHO cell transfectants growingin hygromycin, one cell line was selected based on its high levels ofcyan fluorescence protein expression. These levels remained stable overmultiple generations; genomic DNA blotting verified that a singlechromosomal integration event had occurred. A (loxP—hERG-IBES-neomycinresistance marker—loxP) cassette was subsequently recombined into thisrecipient cell line by CRE recombinase mediated exchange. The correctrecombination event in selected CHO cell clones was verified by i)absence of cyan fluorescence, ii) sensitivity to hygromycin and iii)successful genomic DNA PCR using a forward primer located in the CMVpromoter and a reverse primer in the hERG-ORF. Transfection andsubsequent selection of 5 million cells yielded 16 clones that satisfiedthe above three criteria. These clones were scaled up and analyzed forexpression of hERG protein by western blot and hERG ion channel activityon the Ionworks instrument. One resulting cell line, “CHO crelox hERGUG#7”, was subsequently adapted to growth in suspension. An initialsuspension culture was prepared by diluting cells to 0.75 million/ml inEx-cell 301 medium, 5% FBS, 0.25 mg/ml G418. This culture was grown for24 hrs, and reached a density of about 10⁶ cells/ml. It was then dilutedto 0.2 million cells/ml in fresh medium, and grown for 72 hrs, to reacha density of 10⁶ cells/ml. This dilution-transfer was repeated fourtimes, at which point the cells were considered adapted to suspensionculture. This cell line was deposited to ATCC under the provisions ofthe Budapest Treaty under accession number PTA-6812 on Jun. 22, 2005.

Example 2 Evaluation of Cells Expressing hERG

A description of the cell line using the IonWorks HT instrument has beendocumented: H. Guthrie, et al. (2005). “A Place for High ThroughputElectrophysiology in Cardiac Safety: Generating a Novel hERG Cell Lineand Screening Early with IonWorks HT.” J Biomol Screening (2005)10(8):832-40.

A description of the cell line using the PatchXpress 7000A instrumenthas been documented: L. Guo and H. Guthrie (2005). “The Role ofAutomated Electrophysiology in the Prediction of QT Prolongation.” JPharmacol Toxicol Methods 52(1):123-35.

Example 3 High-Throughput Screening of hERG Activity

The cell line CHO crelox hERG UG#7 exhibited an average currentamplitude of 800 pA, >80% seal rate success, seals between 100-200 MOhmand greater than 80% overall successful recordings, and was chosen to beused in the compound library screen. Three hundred compounds werescreened to find those compounds which inhibited >50% of hERG currentafter a 30 μM compound application. Compounds from several projects wereused for the screen, some of which were already known to block hERGchannels when studied by standard patch clamp, although the experimentshere were performed blinded. Three compound plates were prepared on96-well plates and each was run twice, with approximately 90 compoundsper plate (each compound was tested four times). Utilizing the 384-wellPatchPlate, the 300 compound single-point screen was completed in oneeight-hour day. One run using a compound plate took approximately 40min. In total, each compound was used in eight PatchPlate wells,generally allowing between three and eight successful cells (datapoints) at the one concentration.

During the screen, each compound concentration was screened twice toensure a sufficient number of cells per data point. The redundancy ofeight wells was necessary because of the variability in success rate.Thus is it possible to have a range in n values from one to eight foreach compound. Replicate PatchPlate wells are also important because ofcell-to-cell variability within a cell line. FIG. 2 shows variabilityobserved with the CHO crelox hERG UG#7 cell line over nine experimentaldays (non-consecutive). During this time, after the initial cell lineevaluation, this cell line produced an average current amplitude of 650pA, which was reduced from the 800 pA observed in the cell lineevaluation phase of this study. Current stability was high andapproached 90%. While replicates used in these experiments may haveincreased our screening time, sufficient data was produced within thescreening time designated. With the new IonWorks Quattro, a technologyincorporating Population Patch Clamp™ technology (increased number ofrecording holes in the PatchPlate well), the redundancy practiced herewill be eliminated.

At 30 μM, 160 compounds blocked hERG current >50%. The 160 compounds soidentified were re-screened using an eight-point concentration-responsewith the IonWorks HT system. Five drugs were screened per run, alongwith positive (haloperidol) and negative (PBS with 0.1% DMSO) controls.The average haloperidol IC₅₀ generated with IonWorks HT was 0.74+0.36 μM(from 31 experiments). Historic standard patch clamp data forhaloperidol collected at 37 C with a different cell line under differentexperimental conditions varied from 0.025 to 0.12 μM. The 160 compoundscreen took five days to complete with an individual concentrationtested in 16 PatchPlate wells. As previously discussed, the redundancyin compound testing was to ensure a large number of data points wereproduced to build confidence in the data set. One data set originatingfrom one project was selected for further evaluation since all compoundsin this subset had been tested by standard patch clamp. The historicpatch clamp data and IonWorks HT data were positively correlated(Spearman r=0.53, p<0.004). Compounds that yielded a hERG IC₅₀ value >30μM on IonWorks HT had an average standard patch clamp IC₅₀ value of20.2+10.0 μM (SD). The standard patch clamp values were collected underslightly different conditions including whole cell configuration (versusperforated patch), voltage pulse protocol, recording solutions, cellline (CHO-hERG), temperature (37 C), and analysis methods. Somecompounds appeared to be less potent with the IonWorks HT system, and afew even slightly more potent. However, the average IC₅₀ values of bothgroups showed no statistical difference.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1-3. (canceled)
 4. A method for determining the ability of a testcompound to inhibit hERG, said method comprising: a) providing a stableeukaryotic cell that expresses hERG and exhibits a variation in testcurrent of less than 20% on successive electrophysiological measurementsin a patch clamp apparatus under control conditions, wherein said stableeukaryotic cell comprises CHO crelox-hERG UG#7 (ATCC PTA-68126); b)contacting said cell with a test compound; c) measuring a test currentin a patch clamp apparatus; and d) determining if the test current isreduced in the presence of said test compound.
 5. (canceled)
 6. Themethod of claim 4, wherein said test current in said cell is comparedbefore and after contacting said cell with said test compound.
 7. Themethod of claim 4, wherein said test current in said cell is comparedagainst a test current in a substantially identical cell in the absenceof said test compound.
 8. The method of claim 4, wherein said testcurrent in said cell is compared against a test current in asubstantially identical cell contacted with a compound known to have nohERG-inhibitory effects.
 9. The method of claim 4, wherein said testcurrent in said cell is compared against a test current in asubstantially identical cell contacted with a compound known to exhibitan unacceptable level of hERG inhibition.